Inflammaging is a hallmark of human aging. Defined as low-grade, chronic inflammation, it is characterized by heightened proinflammatory cytokines in the blood and tissues and predicts morbidity and mortality. Despite this, the etiology of inflammaging and its role in infection have remained elusive, an issue this thesis addressed. First, we provided a comprehensive overview of an intranasal Streptococcus pneumoniae colonization model (Chapter 2). We described in detail the colonization technique, and demonstrated how to isolate and phenotype recruited cells, quantify bacterial load and measure production of immune mediators in the nasopharynx. Since both myeloid cell recruitment and tumour necrosis factor (TNF) production were increased following S. pneumoniae colonization with age, we investigated whether TNF directly augmented monocyte frequency (Chapter 3). TNF increased CCR2 expression on monocytes in old mice, leading to their enhanced egress from the bone marrow, resulting in enrichment of this population in the circulation. Monocyte numbers directly influenced plasma IL-6 levels, and this negatively impacted anti-bacterial responses, as monocyte blockade improved pneumococcal clearance in old mice. Lastly, to better understand the fundamental source of inflammaging, we studied the impact of the host microbiome on its development. This work was rooted in Elie Metchnikoff’s early predictions that leakage of intestinal bacterial products could dysregulate macrophage function, resulting in inflammation that would progress aging (Chapter 4). We showed that old mice had increased intestinal permeability, aberrant expression of cellular junction genes and increased microbial translocation from the gut to the blood. Germ-free mice lived longer than their conventionally colonized counterparts, and were protected from the development of inflammaging and defective macrophage function. Together, these studies resolve a major disparity in the field by demonstrating that systemic TNF production is initiated by increased levels of circulating bacterial products, driving functional defects in myeloid cells, which ultimately impairs anti-bacterial immunity. / Thesis / Candidate in Philosophy

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Helper T cells (Th) are a vital component of the immune system responsible for directing other immune cells to eliminate pathogens and cancer. Specifically, Th cells facilitate B cell and cytotoxic T cell (Tc) activation and recruitment and enhance their function against cancer and infectious diseases. Th cells are a valuable resource for improving Tc responses in cancer treatment and have become a focus of immunotherapeutic research. While it is increasingly clear that helper T cells serve an important role, the details about which entities produce an effective Th cell response remain unclear. CD5 is a T cell co-receptor that negatively regulates T cell activation and helps fine-tune the TCR repertoire by altering TCR signaling during the selection process in the thymus. This work discusses the role of the co-receptor CD5 in influencing Th cell metabolism, as well as the study of two T cells called LLO118 and LLO56 that have different CD5 expression levels, and their functional response to altered peptides. Antibodies have revolutionized the world of cancer research and accelerated the development of therapies that trigger the immune system to target disease. In recent years, many antibody-based immunotherapies have emerged as effective candidates for combating cancer due to their refined specificity and ability to target a variety of epitopes. However, many therapies, such as those that target CD19 on B cell cancers, are also present on healthy cells, destroying both cancerous and healthy cells alike. Thymidine kinase 1 (TK1) is an enzyme involved in the DNA salvage pathway that converts thymidine into the nucleotide thymine. Recently, TK1 has been shown to be overexpressed on the surface of many cancers such as acute lymphoblastic leukemia. Importantly, TK1 is not expressed on the surface of healthy cells, making it an ideal cancer-specific antigen that can be targeted for cancer treatment. This work discusses our efforts to characterize TK1-specific single-chain antibodies from a yeast display library. According to the World Health Organization, genomics is defined as the study of all genes and their related functions. In contrast to genetics, genomics analyzes the entire DNA makeup of an organism rather than a single gene. In the past 20 years, the cost of genomic sequencing has decreased dramatically, making it affordable and accessible. A key area that must be addressed with genomic testing involves education about their promise, challenges, potential consequences, and ethical considerations. Genomic testing provides a powerful opportunity to educate everyone on scientific and ethical issues to increase understanding on the subject. This work discusses the influence of personal genomics in society and focuses on the importance, benefits, and consequences of genomics education in the classroom, clinic, and the public.

In humans, various dietary and social factors led to the development of increased brain sizes alongside large adipose tissue stores. Complex reciprocal signaling mechanisms allow for a fine-tuned interaction between the two organs to regulate energy homeostasis of the organism. As an endocrine organ, adipose tissue secretes various hormones, cytokines, and metabolites that signal energy availability to the central nervous system (CNS). Vice versa, the CNS is a critical regulator of adipose tissue function through neural networks that integrate information from the periphery and regulate sympathetic nerve outflow. This review discusses the various reciprocal signaling mechanisms in the CNS and adipose tissue to maintain organismal energy homeostasis. We are focusing on the integration of afferent signals from the periphery in neuronal populations of the mediobasal hypothalamus as well as the efferent signals from the CNS to adipose tissue and its implications for adipose tissue function. Furthermore, we are discussing central mechanisms that fine-tune the immune system in adipose tissue depots and contribute to organ homeostasis. Elucidating this complex signaling network that integrates peripheral signals to generate physiological outputs to maintain the optimal energy balance of the organism is crucial for understanding the pathophysiology of obesity and metabolic diseases such as type 2 diabetes.

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In zebrafish, transgenic labeling approaches, robust regenerative responses and excellent in vivo imaging conditions enable precise characterization of immune cell behavior in response to injury. Here, we monitored osteoblast-immune cell interactions in bone, a tissue which is particularly difficult to in vivo image in tetrapod species. Ablation of individual osteoblasts leads to recruitment of neutrophils and macrophages in varying numbers, depending on the extent of the initial insult, and initiates generation of cathepsin K+ osteoclasts from macrophages. Osteoblast ablation triggers the production of pro-inflammatory cytokines and reactive oxygen species, which are needed for successful macrophage recruitment. Excess glucocorticoid signaling as it occurs during the stress response inhibits macrophage recruitment, maximum speed and changes the macrophage phenotype. Although osteoblast loss is compensated for within a day by contribution of committed osteoblasts, macrophages continue to populate the region. Their presence is required for osteoblasts to fill the lesion site. Our model enables visualization of bone repair after microlesions at single-cell resolution and demonstrates a pro-osteogenic function of tissue-resident macrophages in non-mammalian vertebrates.

According to the centers for disease control and prevention (CDC) and the world healthorganization (WHO), heart disease and immune related diseases such as diabetes and cancer areamong the leading causes of death around the world. Thus, the regulation of the function ofimmune cell plays a key role in health and disease. Calcium (Ca2+) ions play a critical role inimmune cell activation, function and in a robust immune response. Defects in Ca2+ signalinginfluences the development of cardiac disease, Alzheimer disease, immune cell metabolism,muscle dysfunction, and cancer. Each immune cell is unique in its activation and function,making it relevant to understand how activation of each type of immune cell is regulated. Herewe describe the role of the nBMP2 protein in macrophage activation and function and the role ofthe CD5 co-receptor in helper T cell activation and function.The nuclear bone morphogenetic protein 2 (nBMP2) is the nuclear variant of the bonemorphogenetic protein 2 (BMP2), a growth factor important in heart development, neurogenesis,bone, cartilage and muscle development. To better understand the function of nBMP2, transgenicnBMP2 mutant mice were generated. These mice have a slow muscle relaxation and cognitivedeficit caused in part by abnormal Ca2+ mobilization. Mutant nBMP2 mice also have an impairedsecondary immune response to systemic bacterial challenge. Here we have further characterizedmacrophage activation and function from mutant nBMP2 mice before and after bacterialinfection. We describe how nBMP2 influences the Ca2+ mobilization response and phagocytosisin macrophages, revealing a novel role of the nBMP2 protein in immune cell regulation.CD5 is a surface marker on T cells, thymocytes, and the B1 subset of B cells. CD5 isknown to play an important role during thymic development of T cells. CD5 functions as anegative regulator of T cell receptor (TCR) signaling and fine tunes the TCR signaling response.Here we describe our characterization of CD5 regulation of Ca2+ signaling in naïve helper Tcells. We also outline our findings examining how CD5-induced changes in helper T cellactivation influence other biological processes such as immune cell metabolism, the diversity ofthe gut microbiome, and cognitive function and behavior. Thus, this work elucidates theinfluence of the CD5 co-receptor on the functional outcomes in multiple systems when CD5 isaltered.

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